Staphylococcus aureus is an important human pathogen and a reservoir for a large number of plasmids that encode resistance to many commonly used antibiotics. A large group of multicopy plasmids in S. aureus encode resistance to a single antibiotic and replicate by a rolling-circle (RC) mechanism. While several RC plasmids found in S. aureus have a narrow host range, many are able to be stably maintained in a wide range of Gram-positive bacteria. The long term goals of this proposal are to understand the molecular basis for stable replication of RC plasmids in S. aureus and other Gram-positive bacteria, using pTl8l and related plasmids as model systems. We will carry out site-directed mutagenesis to understand the role of ssoA-type origins in lagging strand synthesis of RC plasmids, and the basis of their host-specific function. The molecular basis for broad host range function of the ssoU-type origins will also be investigated. The effect of RepC and the pTl8l origin on the helicase activity of PcrA will be determined. These studies are expected to provide information on the role of this interaction in the initiation and termination of plasmid pTl8l RC replication. The RepC protein acts as a dimer and has DNA binding and nicking-closing domains. Purified heterodimers of wild-type and mutant RepC proteins will be used to identify the role of each monomer during the initiation and termination of pTl8l replication. A new series of experiments utilizing fluorescence microscopy will be initiated to determine whether the pTl81 plasmid replicates at a discrete site in the cell termed the "replication factory." We have obtained co-crystals of RepC bound to its specific binding site and will continue efforts to obtain larger crystals of RepC and RepC-DNA complex and determine their structure by X-ray crystallography. These studies are expected to provide information on the replication and maintenance of drug resistance plasmids in S. aureus and other Gram-positive bacteria that replicate by an RC mechanism.